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Method for generating induced pluripotent stem cells from keratinocytes derived from plucked hair follicles

Inactive Publication Date: 2013-05-23
TECHNION RES & DEV FOUND LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides a method for efficiently generating induced pluripotent stem (iPS) cells from isolated hair follicle keratinocytes. This method involves infecting keratinocytes with a virus containing a nucleic acid molecule encoding at least one dedifferentiation factor, and then culturing the infected keratinocytes on a layer of feeder cells until iPS cells are formed. The iPS cells can be further differentiated into lineage specific cells for research and clinical applications. The invention also provides a pharmaceutical composition comprising the iPS cells and a method for generating lineage specific cells using the iPS cells. The isolated hair follicle keratinocytes can be easily dissociated and infected with the virus, resulting in efficient and reproducible iPS cell generation.

Problems solved by technology

Blood is a cell source that can be easily obtained from most patients, but a practical reprogramming protocol of human peripheral blood cells has not yet been successful.
However, cord blood cannot be obtained directly from most patients, and is therefore an unsuitable source for modeling specific diseases.
However, the reprogramming efficiency obtained with the existing methods does not allow for consistent generation of iPSCs in large numbers required for therapeutic and other uses.

Method used

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  • Method for generating induced pluripotent stem cells from keratinocytes derived from plucked hair follicles
  • Method for generating induced pluripotent stem cells from keratinocytes derived from plucked hair follicles
  • Method for generating induced pluripotent stem cells from keratinocytes derived from plucked hair follicles

Examples

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example 1

The Derivation of Keratinocytes from the ORS of Plucked Hair Follicle and their Characterization

[0168]In order to generate iPSCs from hair, the present inventors followed Aasen et al.'s protocol (Aasen et al. 2008) for generating iPSCs from plucked hair follicles. Plucked hair was cultured on Matrigel-coated dishes supplemented with MEF-conditioned media for at least five days until cells proliferated out of the outer root sheath (ORS), but an insufficient number of viable and proliferative cells could be isolated. The present inventors obtained a single cell suspension of keratinocytes by plucking and selecting at least 10 single hairs with a visible bulb and intact ORS (FIG. 1A), incubating them with DMEM-supplemented with penicillin, streptomycin, HEPES and L-Glu for 24 hr, and then removing the cells from the ORS enzymatically with trypsin-EDTA (FIG. 1B). These isolated keratinocytes were seeded on inactivated 3T3 feeder cells (FIGS. 1C, D) and could be further cultured up to fo...

example 2

Optimization of the Reprogramming Procedure

[0170]With the purpose of developing an efficient HFKT reprogramming protocol, the present inventors used various viral vectors and growth conditions to optimize the reprogramming of common target cells, such as human foreskin fibroblasts (FIFE) and human dermal fibroblasts (HDF). The viral vectors analyzed were the pMX retroviral vectors harboring four (Klf4, Oct3 / 4, Sox2 and c-Myc) or three (Klf4, Oct3 / 4 and Sox2) reprogramming factors (Takahashi et al. 2007; Nakagawa et al. 2008), The pMSCV retroviral vector set modified by Aasen et al. (Aasen et al. 2008) and the humanized version of a single lentiviral STEMCCA vector (SEQ ID NO: 1).

TABLE 5Calibration of post-infection growth-conditionsCellsCellmaintenancesubculturingReprogrammingCell(Fresh / thawGrowthratioEfficiencysourceafter freezing)Viral vectorconditions(post infection)% + SDHFFThaw cellspMX 4FMEF feeder1:60.04% ± 0.03Fresh cells were(OSKM)layernot determinedFibronectin +  1% ± 0.46...

example 3

Induction of Pluripotent Stem Cells from HFKTs, and their Identity Relative to HFKT Parental Cells and hESCs

[0180]The procedure for HFKT reprogramming is illustrated in FIG. 2A. KTN and KTR keratinocytes were derived from plucked hairs of two healthy women, aged 36 and 41, designated “N” and “R”, respectively. Starting with 30,000 keratinocytes which were infected with the STEMCCA vector, ˜5-9 iPSC colonies were isolated in five independent experiments. Importantly, all the colonies that emerged following ˜30 days of incubation were true iPSCs exhibiting morphological features resembling those of hESCs (FIG. 2B). All the iPSC colonies were picked mechanically and were transferred to the MEF feeder-layer for further expansion and analysis. Their karyotypes were analyzed and were found to be normal excluding two clones, one with a lost X chromosome (45, X0) and the other with an unstable karyotype (data not shown).

[0181]Specific characterization of the HFKT-iPSCs, as compared with the...

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Abstract

A method for generating induced pluripotent stem (iPS) cells from isolated hair follicles is disclosed. The method comprises:a. culturing isolated hair follicle keratinocytes on a layer of feeder cells, so as to generate colonies of hair follicle keratinocytes;b. detaching the colonies of hair follicle keratinocytes from the feeder cells so as to generate detached keratinocytes;c. infecting the detached keratinocytes with a virus comprising a nucleic acid molecule encoding at least one dedifferentiation factor so as to generated infected keratinocytes; andd. culturing the infected keratinocytes on a layer of feeder cells in a culture medium until iPS cells are formed, thereby generating iPS cells.Populations and uses of the iPS cells are also disclosed.

Description

TECHNICAL FIELD[0001]The present invention relates to the field of pluripotent stem cell generation from somatic cells and in particular from keratinocytes derived from plucked hair follicle. The invention further relates to therapeutic use of the pluripotent stem cells and to their use in drug screening and disease modeling.BACKGROUND ART[0002]Induced pluripotent stem cells (iPSCs) are human somatic cells that were reprogrammed into a pluripotent state resembling that of human embryonic stem cells (hESCs). iPSCs are generated by introducing a defined set of transcription factors, including Oct4 (Octamer-4, also known as POU5F1), Sox2 (SRY (sex determining region Y)-box 2), Klf4 (Krüppel-like factor 4) and c-Myc or Nanog and Lin 28 (Takahashi et al. 2007; Yu et al. 2007). The seminal achievement of induced pluripotency holds great promise for regenerative medicine. Patient-specific iPSCs can provide useful platforms for the discovery of new drugs, as well as unprecedented insights i...

Claims

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Application Information

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IPC IPC(8): C12N15/85
CPCC12N5/0696C12N2501/602C12N2501/603C12N2501/604C12N2501/606C12N15/85C12N2501/727C12N2506/092C12N2506/094C12N2510/00C12N2501/72A61P1/16A61P25/16A61P37/00A61P43/00A61P9/10A61P3/10
Inventor ITSKOVITZ-ELDOR, JOSEPHNOVAK-PETRARO, ATARASHTRICHMAN, RONIT
Owner TECHNION RES & DEV FOUND LTD
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